Glycosaminoglycan modification of neuropilin-1 modulates VEGFR2 signaling - PubMed (original) (raw)

. 2006 Jul 12;25(13):3045-55.

doi: 10.1038/sj.emboj.7601188. Epub 2006 Jun 8.

Seiji Takashima, Yoshihiro Asano, Hisakazu Kato, Yulin Liao, Satoru Yamazaki, Osamu Tsukamoto, Osamu Seguchi, Hiroyuki Yamamoto, Tomi Fukushima, Kazuyuki Sugahara, Masafumi Kitakaze, Masatsugu Hori

Affiliations

Glycosaminoglycan modification of neuropilin-1 modulates VEGFR2 signaling

Yasunori Shintani et al. EMBO J. 2006.

Abstract

Neuropilin-1 (NRP1) is a co-receptor for vascular endothelial growth factor (VEGF) that enhances the angiogenic signals cooperatively with VEGFR2. VEGF signaling is essential for physiological and pathological angiogenesis through its effects on vascular endothelial cells (ECs) and smooth muscle cells (SMCs), but the mechanisms coordinating this response are not well understood. Here we show that a substantial fraction of NRP1 is proteoglycan modified with either heparan sulfate or chondroitin sulfate on a single conserved Ser. The composition of the NRP1 glycosaminoglycan (GAG) chains differs between ECs and SMCs. Glycosylation increased VEGF binding in both cell types, but the differential GAG composition of NRP1 mediates opposite responsiveness to VEGF in ECs and SMCs. Finally, NRP1 expression and its GAG modification post-transcriptionally regulate VEGFR2 protein expression. These findings indicate that GAG modification of NRP1 plays a critical role in modulating VEGF signaling, and may provide new insights into physiological and pathological angiogenesis.

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Figures

Figure 1

Figure 1

A substantial fraction of cellular NRP1 is proteoglycan, composed of either HS or CS. (A) 125I-labeled VEGF is crosslinked to different proteins in ECs and SMCs. Arrow indicates VEGF-binding protein specifically seen in SMCs. CASMC: coronary artery smooth muscle cell; BSMC: bronchial smooth muscle cell; HUVEC: human umbilical vein endothelial cell. (B) Western blots of exogenously expressed NRP1 in either CASMCs or HUVECs. Adenovirus encoding FLAG-tagged NRP1 was transfected 2 days before analysis at the indicated MOI. LacZ-encoding adenovirus was used as a control. (C) The high molecular weight band was not simply a covalently linked homodimer of NRP1. Only FLAG-tagged NRP1 or both FLAG-tagged and V5-tagged NRP1 were transfected in CASMCs, and the cell lysates were immunoprecipitated and detected by the indicated antibody. (D) Endogenous NRP1 was modified by GAG chain addition in both SMCs and ECs. The upper band in CASMC immunoprecipitates disappeared following treatment with both HSase and CSase. HUVEC-expressed NRP1 is also GAG modified. The band intensity was analyzed and the proportion of each glycanated form of NRP1 was determined. Data are from three separate experiments. HSase: heparitinase; CSase: chondroitinase.

Figure 2

Figure 2

(A) NRP1 is GAG modified on a single Ser612 residue. CASMCs were transfected with adenoviral vectors encoding WT or S612A mutant NRP1. NRP1 S612A is not GAG modified. (B) Multiple alignments of NRP1 from different species. Ser612 is highly conserved among vertebrates. (C) NRP2, an NRP family member, is not GAG modified. (D) Design of siRNA and adenovirus constructs. Ser612 exists in the bridge region between the b1b2 and MAM domains. (E) Replacement of Ser612 by Ala612 of NRP1 did not change binding to VEGF. Cos7 cells were transfected with either NRP1 WT′ or S612A expression vector and preincubated with heparitinase (1.5 mU/ml), heparinase (1.5 mU/ml), and chondroitinase (20 mU/ml) in the culture medium at 37°C for 2 h to make NRP1 non-GAG form. After incubation with 125I-labeled VEGF for 30 min at room temperature, cell lysates were immunoprecipitated by anti-NRP1 antibody, and the bound radioactivity was quantitated using a gamma counter. Data are from three independent experiments. For panel E, error bars represent s.e.

Figure 3

Figure 3

GAG modifications differentially affect NRP1 function in SMCs and ECs. (A) Experimental replacement of NRP1 in SMCs and ECs. After transfection with both N-G siRNA and adenoviral constructs, endogenous NRP1 was successfully replaced with either the glycanated form (NRP1 WT′) or non-glycanated form (NRP1 S612A) of NRP1. Tubulin was used as a loading control. (B) Addition of GAG to NRP1 enhances binding to VEGF in both types of cells. Two days after NRP1 replacement, cell lysates were immunoprecipitated with anti-FLAG antibody after incubation with 125I-labeled VEGF (25 ng/ml) for 40 min at room temperature, and bound radioactivity was quantitated using a gamma counter. Heparitinase and chondroitinase treatment with these immunoprecipitates could not entirely eliminate the enhancement of VEGF binding. Data are from three independent experiments. (C) VEGF (50 ng/ml) induced greater cell migration in SMCs expressing non-modified NRP1 S612A than those expressing NRP1 WT′. Migrated cells were quantified by counting cells in three random high-power fields (HPF, × 200). Similar results were obtained from additional two independent experiments. (D) VEGF (50 ng/ml) increased cell viability in ECs expressing NRP1 WT′ to a greater extent than in ECs expressing NRP1 S612A. Data are from three independent experiments. For panels B–D, error bars represent s.e. *P<0.05, versus adeno-NRP1 WT′ in panel B.

Figure 4

Figure 4

Different roles of the GAG of NRP1 on VEGFR2 in SMCs. (A) Experimental replacement with NRP1 S612A increased VEGFR2 expression in SMCs, but replacement did not affect VEGFR2 expression in ECs. Two days after transfection with siRNA and adeno-NRP1, cells were analyzed by Western blotting. Data are representative of at least three independent experiments. (B) Quantitative results of Western blot. (C) Experimental replacement with NRP1 S612A increased VEGFR2 protein expression without any transcriptional change in SMCs. Each sample was analyzed in duplicate and the experiments were performed in triplicate for the full set of genes. (D) CS-modified NRP1 had the same affinity for VEGF as HS-modified NRP1 and non-modified NRP1. Note that 125I-labeled VEGF bound CS-modified NRP1 with a similar ratio before crosslink (upper band in lanes 4 and 8, CS-modified NRP1:HS-modified NRP1=2:1). Increasing amounts of cold VEGF equally inhibited 125I-labeled VEGF binding to all forms of NRP1. (E) Co-immunoprecipitation of NRP1 with VEGFR2. CS-modified NRP1 (left panel, about 250 kDa in lane 4) minimally associated with VEGFR2 compared to non-modified (130 kDa, in lanes 2, 4, 6, 8) and HS-modified NRP1 (about 250 kDa in lane 6), although there was a two-fold excess of CS-modified NRP1 compared to HS-modified NRP1 at input (right panel). The membrane was stripped and re-probed with anti-V5 as a loading control. Data are representative of at least three independent experiments. (F) The rate of VEGFR2 degradation was decreased in NRP1 WT′ ECs compared to NRP1 S612A ECs. Phosphorylated VEGFR2 was also much higher in NRP1 WT′ ECs than in NRP1 S612A ECs at any time point after VEGF. Data are representative of at least three independent experiments. For panels B, C, F, error bars represent s.e. *P<0.05, versus NG/ NRP1 S612A at the same period as in panel F. HSase: heparitinase; CSase: chondroitinase.

Figure 5

Figure 5

NRP1 post-transcriptionally regulates the expression of VEGFR2. (A) Both NRP1 siRNAs (N-G, N-1) decreased VEGFR2 expression. In contrast, VEGFR1 was not influenced by NRP1 knockdown. Tubulin was used as a loading control. Data are representative of at least three independent experiments. (B) Transcription levels of both VEGFR1 and VEGFR2 were not influenced by NRP1 knockdown. Each sample was analyzed in duplicate and experiments were performed in triplicate for the full set of genes. (C) Pulse–chase experiments in HUVECs. The rate of degradation of VEGFR2 was not changed by NRP1 knockdown. Data are from four independent experiments. (D) NRP1 significantly upregulated VEGFR2 protein levels in Flp293/VEGFR2 cells. Transfected NRP1 in Flp293/VEGFR2 cells was GAG modified similar to ECs. Data are representative of two independent experiments. (E) NRP1 regulates cell-surface VEGFR2 expression. Transient expression of FLAG-tagged NRP1 WT upregulated the cell membrane-associated VEGFR2 expression compared to adjacent non-transfected cells. Flp293/VEGFR2 cells were transfected with either NRP1 WT or mock and stained without permeabilization using anti-VEGFR2 (green) and anti-FLAG-Cy3 (red). Blue: DAPI nuclear staining. For panels A and C, numeric represents the mean of band intensity of three experiments. For panel B, error bars represent s.e.

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